C-KIT (also known as KIT, Cluster of Differentiation 117 (CD117), PBT, SCFR, KIT proto-oncogene receptor tyrosine kinase) is a transmembrane protein that belongs to the immunoglobulin superfamily and binds to the soluble factor SCF (stem cell factor). C-KIT is a receptor tyrosine kinase type III that is highly expressed by hematopoietic stem cells as well as multiple other cell types, such as mature Mast Cells, where SCF signalling acts as a cytokine. On binding to SCF, this receptor dimerises, activating its tyrosine kinase activity. This kinase activation leads to further downstream activation of signal transduction molecules that play known roles in cell survival, proliferation, and differentiation.
Altered forms of C-KIT, such as constitutively active mutants, are strongly associated with the progression of several important types of cancer, such as Gastrointestinal Stromal Tumours (GIST), Acute Myeloid Leukaemia (AML), Mast Cell tumours and Melanoma. Preclinical and clinical evidence suggests that blocking C-KIT-SCF signalling can have clear therapeutic benefit in multiple cancers, but this has predominantly been achieved using small molecule inhibitors of C-KIT kinase function. Resistance mutations commonly develop after treatment, causing the therapeutic efficacy of the small molecule kinase inhibitor to be lost. Therapeutic antibodies that antagonise KIT signalling by blocking the ability of the receptors to dimerise have the potential to overcome kinase inhibitor resistance and mediate anti-tumour effects, via two mechanisms: 1. Potent inhibition of the KIT signalling pathway by locking the receptors into a non-activating monomeric form. 2. Antibody effector-function mediated engagement of immune cells. Importantly, it has recently been recognised in preclinical studies that C-KIT-SCF signalling in mast cells found in the tumour microenvironment (via SCF produced by stromal cells), can promote downstream cytokine signalling that recruits myeloid cells such as Myeloid-Derived Suppressor Cells (MDSCs). As MDSCs are believed to be a key cell population that suppress immune responses against tumours, the indirect inhibition of their tumour infiltration via KIT signalling antagonism may be an attractive therapeutic strategy.
The majority of currently approved antibody therapeutics are derived from immunized rodents. Many of those antibodies have undergone a process known as “humanization”, via the “grafting” of murine Complementarity-Determining Regions (CDRs) into human v-gene framework sequences (see Nelson et al., 2010, Nat Rev Drug Discov 9: 767-774). This process is often inaccurate and leads to a reduction in target binding affinity of the resulting antibody. To return the binding affinity of the original antibody, murine residues are usually introduced at key positions in the variable domain frameworks of the grafted v-domains (also known as “back-mutations”).
While antibodies humanized via CDR grafting and back mutations have been shown to induce lower immune response rates in the clinic in comparison to those with fully murine v-domains, antibodies humanized using this basic grafting method still carry significant clinical development risks due to the potential physical instability and immunogenicity motifs still housed in the grafted CDR loops. As animal testing of protein immunogenicity is often non-predictive of immune responses in man, antibody engineering for therapeutic use focuses on minimizing predicted human T-cell epitope content, non-human germline amino acid content and aggregation potential in the purified protein.
The ideal humanized antagonistic anti-C-KIT antibody would therefore have as many residues as possible in the v-domains that are identical to those found in both the frameworks and CDRs of well-characterized human germline sequences. This high level of identity to high-stability germlines that are highly expressed in the maximum number of potential patients minimises the risk of a therapeutic antibody having unwanted immunogenicity in the clinic, or unusually high ‘cost of goods’ in manufacturing.
Townsend et al. (2015; PNAS 112: 15354-15359) describe a method for generating antibodies in which CDRs derived from rat, rabbit and mouse antibodies were grafted into preferred human frameworks and then subject to a human germ-lining approach termed “Augmented Binary Substitution”. Although the approach demonstrated a fundamental plasticity in the original antibody paratopes, in the absence of highly accurate antibody-antigen co-crystal structural data, it is still not possible to reliably predict which individual residues in the CDR loops of any given antibody can be converted to human germline, and in what combination. Additionally, the Townsend et al. study did not address the addition of mutagenesis beyond the residues found in the human germline at positions where the removal of development risk motifs might be beneficial. This is a technological limitation which renders the process inherently unsatisfactory as it allows the retention of development liability motifs in CDRs.
CDR germ-lining is thus a complex, multifactorial problem, as multiple functional properties of the molecule should preferably be maintained, including in this instance: target binding specificity, affinity to C-KIT from both human and animal test species (e.g. cynomolgus monkey, also known as the crab-eating macaque, i.e. Macaca fascicularis), v-domain biophysical stability and/or IgG yield from protein expression platforms used in research, clinical and commercial supply. Antibody engineering studies have shown that mutation of even single residue positions in key CDRs can have dramatic effects on all of these desired molecular properties.
WO2014018625A1 describes an antagonistic murine anti-C-KIT IgG molecule termed “37M”, and also the preparation of humanized forms of 37M. Those humanized forms of 37M were produced using classical humanization techniques, i.e. by grafting of Kabat-defined murine CDRs into human heavy and light chain framework sequences, with some of the human framework residues being potentially back-mutated to the correspondingly positioned 37M murine residues. For reasons noted above, such humanized forms of 37M described in WO2014018625A1 are not ideal.
The present invention provides a number of optimized anti-C-KIT antibodies and medical uses thereof.